[CANCER 57.466-471. February1, 1997]
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Herpes Virus to Treat Experimental
Use of a â€œReplication-Restrictedâ€• Human
John C. Kucharczuk,' Bruce Randazzo,' Michael Y. Chang, Kunjiata M. Amin, Ashraf A. Elshami,
Daniel H. Sterman, Nabil P. Rizk, Katherine L. Molnar-Kimber, S. Moira Brown, Alasdair R. MacLean,
Leslie A. Litzky, Nigel W. Fraser, Steven M. Albelda, and Larry R. Kaiser@
Thoracic Oncology Research Laboratory Ii. C. K.. M. Y. C., K. M. A., A. A. E., D. H. S.. N. P. R.. K. L M-K.. L A. L. S. M. A., L R. K.] and Department of Dermatology [B. R.].
University of Pennsylvania Medical Center, Philadelphia, Pennsylvania 19104; Wistar Institute, Philadelphia, Pennsylvania 19104 (B. R., N. W. F., S. M. A.]: and Glasgow
University, Neurovirology Research Laboratories, Glasgow, Scotland 651 4TF (S. M. B., A. R. M.]
ABSTRACT (12). This mutation, through mechanisms that are still unclear, se
verely attenuates the ability of HSV-l716 to replicate in normal
MOdified, nonneurovirulent herpes simplex viruses (HSVs) have shown
tissues (12, 17, 18), but does not appear to affect the virus' ability to
promise in the treatment of brain tumors. However, HSV-1 can infect and
replicate in rapidly dividing malignant cells. We and others (18, 19)
lyse a wide range of cell types. In this report, we show that HSV-1716, a
mutant lacking both copies of the gene coding ICP-34.5, can effectively
have shown that HSV-1716 is extremely nonneurovirulent in animal
treat a localized Lp malignancy. Human malignant mesothelloma cells models and can be used to treat intracranial tumors in normal and
supported the growth of HSV-1716 and were efficiently lysed in vitro. i.p. immunodeficient mouse models (15, 16, 20).
injection of HSV-1716 into animals with established tumor nodules re Neurovirulent, wild-type HSV-l is able to infect and lyse a wide
duced tumor burden and significantly prolonged survival in an animal variety of cell types. Therefore, we reasoned that a mutant HSV virus,
model ofnon-central nervous system-localized human malignancy without such as HSV-17l6, might be efficacious in the treatment of localized,
dissemination or persistence after i.p. injection Into SC@ mice bearing non-CNS malignancies. In addition, the use of this virus in a location
human tumors. These findings suggest that this virus may be efficacious distant from the CNS might offer significant safety advantages. Ac
and safe for use in localized human malignancies of noaneuronal origin cordingly, in this paper we report on the successful in vitro and in vivo
such as malignant mesothelioma.
use of HSV-l716 to treat a localized, non-CNS malignancy. We have
chosen human malignant mesothelioma as a model tumor system for
INTRODUCTION HSV-l716 because of the localized nature of this malignancy (allow
ing for direct virus administration) and because of the current lack of
The recent emergence of viral-based â€œgene therapyâ€•as an approach effective treatment (21).
to treat cancer has generated a great deal of enthusiasm and interest
(1). For example, our group and others have explored the use of
MATERIALS AND METHODS
retrovirus-producing cells or adenoviruses to deliver the HSV& gene
into localized malignancies, such as brain tumors and malignant HSV-1716. HSV-l716 was originally isolated in the laboratory of S. M.
mesothelioma (2â€”6).Although a â€œbystanderâ€• effect amplifies the Brown(Glasgow,Scotland)andpassagedfor use in this studyby N. W. Fraser
efficacy of the HSVtk/ganciclovir system (7), it is likely that a major (Philadelphia, PA). The genome of this virus contains a 759-bp deletion
obstacle in treating actual patients with localized malignancies will be located within each copy of the BamHI fragment of the long repeat region of
the ability to transduce a sufficient number of cells within a large the genome (12). These deletions remove most of the gene encoding ICP 34.5,
tumor mass. One strategy that holds promise is the use of replicating and the mutant fails to make the protein (12, 22)
In Vitro Studies of HSV-1716 on a Human Malignant Mesothelioma
viral vectors (8).
Cell Line. A humanmalignantmesotheliomacell line call REN was isolated,
The idea of using replicating viruses to treat tumors was suggested characterized, and passaged as described previously by our laboratory (23).
as early as 1904 by George Dock (9); in fact, a number of clinical Human lung cancer cell lines A549, H322, and H358 were obtained from the
trials were conducted in the l950s and 1960s with some success (10, American Type Culture Collection (Rockville, MD). The human osteosarcoma
11), although interest in the use of viral-based cancer therapy waned cell line SAOS was obtained from T. Halazonetis (The Wistar Institute). The
thereafter. Advances in virology and molecular biology now allow the human melanoma cell line WM451 Lu was obtained from M. Herlyn (The
â€œengineeringâ€•of viruses with specific properties, suggesting that the Wistar Institute) and was isolated, characterized, and passaged as described
idea of viral-based cancer therapy should be revisited. One promising previously (24). To construct single-step viral growth curves, cells were plated
virus in this regard is HSV-l. A number of HSV-l mutants have on six-well plates at a density of 5 X iO@cells/well and infected 24 h later with
HSV-l7l6 at a MO! of 0.01 (5 X l0@PFU/well). One well was harvested at
recently been identified that appear to replicate preferentially in
0, 6, 12, and 24 h by cell scraping and collection of the media. The samples
rapidly dividing transformed cells (12, 13). Because of the natural
were freeze/thawed and titered by plaque assay on baby hamster kidney cell
tropism of wild-type herpes virus for neuronal tissue, the published monolayers. A cell viability assay was performed by plating cells in 96-well
uses of modified, replicating HSV to treat cancer have centered on plates at a density of 5 X l0@ cells per well. Twenty-four h later, the cells were
tumors of CNS origin (14â€”16).One of these mutants is HSV-1716, a infected with HSV-l716 at MOIs of 0, 0.001, 0.01, and 0.1. Six wells were
virus that contains a 759-bp deletion in the genes coding for ICP 34.5 infected at each MO!. A sufficient number of plates were used to allow for
viability assay at 24, 48, 72, and 96 h after infection. Viable cell number was
Received 7/29/96; accepted 12/4/96. assessed by a colorimetric assay (CellTiter 96 Aqueous Nonradioactive MTT
The costs of publication of this article were defrayed in part by the payment of page Cell Proliferation Assay; Promega, Madison WI) that measures viable cell
charges. This article must therefore be hereby marked advertisement in accordance with dehydrogenase activity by absorbance. The percentage of control growth is
18 U.S.C. Section 1734 solely to indicate this fact.
defined as the ratio of the mean absorbance of six treatment wells at 490 nm
I The first two authors contributed equally to this work.
2 To whom requests for reprints should be addressed, at Division of Thoracic Surgery, to the mean absorbance of six untreated matched controls.
University of Pennsylvania Medical Center, 4 Silverstein, 3400 Spruce Street, Philadel In Vivo Studies. A previously described model of human malignant me
phia, PA 19104. Phone: (215) 662-7538. sothelioma growing in the peritoneal cavity of SCID mice was used for all in
3 The abbreviations used are: HSVtk, herpes simplex thymidine kinase; HSV-l, herpes
vivoexperimentation (23). Animal protocols were approved by the Animal Use
simplex-l virus; CNS, central nervous system; ICP, infected cell protein; MO!, multi
plicity of infection; PFU, plaque-forming unit; MT1', 3-(4,5-dimethylthiazol-2-yl)-2,5 committees of the Wistar Institute and the University of Pennsylvania in
diphenyltetrazolium bromide. compliance with the Guide for the Care and Use of Laboratory Animals (NIH
TREATMENT MALIGNANT WITh HS@
No. 85-23, revised 1985). Briefly, SCID mice were obtained and housed at
the animal facilities of the Wistar Institute. On day 0, animals were injected
i.p. with 3 X l0@ REN cells in 1 cc of cell culture media. For the tumor
burden study, treatment animals were given 5 X 106PFU of HSV-1716 in
culture media by i.p. injection on day 14. Control animals received an
equivalent volume of culture media. The animals were examined daily and
sacrificed by cervical dislocation on day 28. The amount of tumor burden ioe
was assessed using a four-point semiquantitative scale, which accounts for
both gross and microscopic disease. Briefly, animals were assessed for
tumor in the following four areas: stomach/pancreas, portal region, retro
peritoneum/diaphragm, and small bowel mesentery. On gross examination,
animals received either a score of 0 if no tumor was present or a score of
1 in each of the four designated areas where gross tumor was seen. If no
gross tumor was visible, H&E-stained paraffin-embedded sections of each @o ::
organ from the designated area were examined in a blinded fashion by an
anatomic pathologist. The sections were scored as either 0 for no micro
@ scopic tumor or 0.5 if microscopic tumor was present. Thus, the tumor io2
scores ranged from 0 to 4.0. Organs including brain, heart, lungs, liver,
stomach, pancreas, kidney, adrenals, spleen, gonads, small bowel, and 10
diaphragm were obtained from each animal. Each organ was divided into
thirds with equal samples designated for frozen section, formalin fixation, 100
and DNA extraction. 0 6 12 24
For the initial survival study, animals were injected i.p. with 3 X l0@REN (hours)
cells in 1 ml of cell culture media (day 0). On day 7, one animal was sacrificed
for gross tumor confirmation, and the remaining animals were randomized to B
the treatment group and the control group. Treatment animals received 5 X 106 100
PFU of HSV-l7l6 by i.p. injection; control animals received an equal volume
of culture media. The animals were checked daily and followed for survival.
The same protocol was followed for the dose-response study, except the
animals were randomized into a control group, a high dose group (5 X 106 80
PR! HSV-l716), a middle dose group(5 X l0@' PFU HSV-l7l6), and a low
dose group (5 X 10@ PFU HSV-l7l6). A similar protocol was followed to -Kâ€” M.O.I. = 0.0
evaluate the early deaths observed in the survival studies, except that the -.- M.O.I. = 0.01
60 -A- M.O.$. = 0.1
tumor-bearing animals were sacrificed 3 days after viral administration. Blood
-.- M.O.I. -1
samples were obtained, pooled, and evaluated for serum creatinine and serum .0
uric acid level. Serum chemistry analysis was performed in the Pepper Clinical >
Laboratory at the University of Pennsylvania Medical Center (Philadelphia, 40
Histology and Immunohistochemistry. As reported previously, the REN
cell line was isolated from a patient with the appropriate clinical and patho
logical features of malignant mesothelioma, including morphological and 20
immunohistochemical staining pattern (CEA, LeuMl, and mucicarmine anti
body negative; Ref. 23). To further assure the nonneuronal origin of these cells
for the purposes of this study, REN-derived tumors were stained immunohis
tochemically with an antibody called TA-5l (a gift of J. Q. Trojanowski, 24 48 72 96
University of Pennsylvania, Philadelphia, PA), which recognizes neurofila (Hours)
ment H, a specific marker for neuronal lineage (25). REN cells were TA-S1
negative (data not shown). Immunohistochemical staining for HSV antigens Fig. 1.A, HSV-17l6 single-step viral growth curve on human malignant mesotheioma
cells. Inoculum at time 0 was 5000 PR] of virus (MOl = 0.01). Note that at 24 h the
was performed on paraffin sections of tissues fixed in 10% phosphate-buffered amount of virus present had increased by fourlogs over the initial input inoculum. B, MU
formalin, using a commercially available rabbit anti-HSV polyclonal antibody assay for human malignant mesotheioma cell viability as a function of time and varying
(DAKO, Carpinteria, A).
C o i cells
MOl.Thepercentage fcontrolsurvival stheratioofmeanMTI'activityininfected
(n 6 wells at each time point) to the mean activity in matched uninfected cells (n = 6
In Vivo Dissemination and Restriction Studles To look for dissemina wells at each time point).
tion of HSV-l7l6, we performed PCR looking for the HSVtk gene in the
collected tissues. Genomic DNA was obtained by standard phenol/chloro
form extraction and amplified by PCR. The PCR primers (sense ATG
GCTITFCGTACCCCTGCCAT and antisense GGTATCGCGC0000G- RESULTS
GGTA) were designed to span a region of the HSVtk gene generating a
HSV-1716 Efficiently Replicates in a Human Malignant Me
536-bp fragment. The genomic DNA extracted from each tissue sample was
subjected to 35 cycles of PCR using the tk primers. The tk plasmid and sothelioma Cell Line and Lyses the Cells in Vitro. To determine the
DNA extracted from brain tissues of an animals infected with wild-type ability of HSV-1716 to replicate within a non-CNS human tumor cell
HSV- 17+ were used as positive controls for the PCR reaction. PCR line in vitro, we performed a single-step viral growth curve in REN
products were run on ethidium bromide containing 1.5% agarose gels, and cells. As shown in Fig. 1A, REN cells supported efficient growth of
the positives produced the expected 536-bp fragment. The gels were then 1716. Immediately after infection (time 0), 70% of the input viral
overnight blotted onto Zeta-Probe GT blotting membranes (Bio-Rad Lab inoculum was recovered. By 6 h, the number of recovered active viral
oratories, Hercules, CA). The membrane was UV cross-linked and probed particles fell by a factor of 200, as expected due to viral uptake and
using a 32P-Iabeled portion of the HSVtk plasmid. disassembly in preparation for viral replication. However, at 24 h, a
@ I ,@A. 0@ @. .@ . V
TREATMENT MALIGNANT HSV
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Fig. 2. Immunohistochemistry for HSV viral proteins in SC@ mice. Micrographs 3â€”5 after i.p. injection of wild-type virus HSV-17+ . Positive immunostaining (brown cells)
is seen in the normal tissues, including ganglia of the myenteric plexus (a), adrenal gland (b), and brain (c). In contrast, d shows a tumor-bearing animal 72 h after receiving 5 X l0@
PFU of HSV-17l6 by i.p. injection. Note the marked positivity of tumor cells with the lack of infection in the surrounding normal tissues (X200, arrowheads mark the interface between
tumor (1) and normal tissues).
4-log increase over the initial inoculum was observed demonstrating mcd, the human lung cancer lines A549 and H322, were lysed poorly
highly efficient replication of HSV-1716 on REN cells. by both HSV-l716 and wild-type HSV-l7+ (data not shown).
To demonstrate the ability of HSV-17l6 to directly kill REN cells, In Contrast to Wild-Type HSV-17+, HSV-1716 Infection and
we performed an in vitro cell viability assay at various times after Replication Is Restricted to Tumor Cells in an in Vivo SC@
viral infection. As shown in Fig. lB. HSV-1716 efficiently killed Mouse Model of Human Mesothelioma. As expected, i.p. injection
target REN cells in a time- and dose-responsive fashion. At 96 h of SCID mice with 5 X 106 PFU of wild-type HSV-17+ led to rapid
postinfection, there were no viable tumor cells remaining in cultures spread of the virus, signs of encephalitis (i.e., ataxia and hind limb
infected at MOIs ranging from 0.01 to 1.0. Similar results were paralysis), and death of all animals by 7 days. To determine the extent
obtained for several other nonneuronal cell lines tested in this viability of HSV infection, organs from animals sacrificed 3â€”5 days after
assay, such as the human osteosarcoma line SAOS, the human lung wild-type HSV-HSV-17+ injection were analyzed immunohisto
cancer line H358, and the human melanoma cell line WM 45 1 Lu chemically with a polyclonal antibody recognizing HSV antigens.
(data not shown). However, two other nonneuronal cell lines exam Reactive cells were clearly seen in the myenteric ganglia of the small
OF MESOTHELIOMA ITHHSV
TREATMENT MALIGNANT W
intestine (Fig. 2A), adrenal glands (Fig. 2B), and brain (Fig. 2C). In period. There was one death in the treatment group that occurred 5
contrast to wild-type HSV-HSV-l7@, SCID mice (n = 5) injected days after viral administration from unknown cause.
with 5 X 106 PR] of HSV-l716 remained alive at 100 days without To determine whether the decrease in tumor mass conferred a
signs of encephalitis. Immunohistochemistry for HSV antigens was survival advantage to SCID mice bearing established i.p. REN tumors,
negative at days 3, 8, and 14 in all tissues analyzed. Specifically, liver, a group of tumor-bearing animals were injected with 5 X 106 pp@j of
kidney, adrenal, spleen, small bowel myenteric plexuses, and brain HSV-17l6 2 weeks after i.p. injection of tumor cells. The animals
were negative for HSV antigen. were followed for survival. The median survival was increased from
To assess the ability of HSV-17l6 to infect and replicate within 47 days in the control group (n = 9) to 95 days in the treatment group
human tumors in vivo, SCID mice were injected i.p. with 30 million (n 10). All deaths in the control group were a result of bulky i.p.
human REN cells. After 14 days, diffuse macroscopic 5â€”8-mm tumor disease; no external tumor nodules were visible at the initial tumor
nodules were present. At this time, 5 X 106 PFU of HSV-17l6 were injection site. It is interesting that deaths in the treatment group
instilled into the peritoneal cavity, and the animals were sacrificed d
occurred at two distinct time points. Three animals died 2â€”5ays after
72 h later. Microscopic examination revealed that virtually all tumor HSV-1716 administration. There was no evidence of bulky disease at
this time. The majority of the remaining treated animals died around
nodules showed necrosis, multinucleated cells, and nuclear inclusions
day 100 due to bulky malignant disease that extended from large s.c.
consistent with active herpetic infection. Infiltration with mononu
nodules arising on the anterior abdominal wall. At 102 days, the three
clear inflammatory cells was also present. In contrast, no viral cyto
remaining treatment animals were sacrificed and necropsied. These
pathic changes were seen in normal tissues. To directly detect HSV
animals also had nodules corresponding to the site of the initial tumor
infection, tumor tissue and organs were stained with an anti-HSV
injection, and tumor appeared to be growing inward from the anterior
polyclonal antibody. Fig. 2D shows a representative tumor nodule
abdominal injection site with invasion into the peritoneal cavity.
growing on the inferior aspect of a SCID mouse stomach. Three days
A second survival study was performed to determine the viral dose
after HSV-l716 administration, a large percentage of the tumor cells
response. Tumor-bearing animals were randomized to control
stained positively for HSV antigens, whereas surrounding normal (n 11) and treatment groups (low dose: 5 X l0@ PHi HSV-l716,
tissues, as well as all other normal tissues examined, showed no n 10; middle dose: 5 X l0@ PHi HSV-l7l6, n 10; high dose:
positive staining. Specifically, liver, kidney, adrenal, spleen, small 5 x 106 PHi HSV-l716, n 10). As shown in Fig. 4, treatment with
bowel myenteric plexuses, and brain were negative for HSV antigen. high dose HSV-17l6 significantly improved survival when compared
Similar results were obtained at days 5, 7, and 14 after infection; with control animals (P = 0.001 1 by Mantel-Cox log-rank test).
however, the number of virally infected tumor cells appeared to Animals in the high dose group had a mean survival of 102 days;
decrease at the later time points, possibly due to a decrease in again, surviving animals developed s.c. tumor nodules on the anterior
available tumor substrate. abdominal wall corresponding to the initial tumor injection site by day
To more sensitively monitor the dissemination of HSV-1716 in 100. Six of these animals subsequently died from bulky tumor ex
tumor-bearing animals, we used the PCR followed by Southern blot tending from the anterior abdominal wall injection site into the peri
ting to detect the HSVtk gene. This assay can detect HSV DNA at a toneal cavity. The low and middle dose treatment animals also dem
level of sensitivity equivalent to one genome copy per 100,000 cells.4 onstrated a significant improvement in survival when compared with
Fig. 3A shows the results from a tumor-bearing mouse 3 days after i.p. the control animals (P = 0.0003 for control versus middle dose and
installation of 5 X 106 PFU of HSV-l 716. HSV DNA was detected in P = 0.0019 for control versus low dose by Mantel-Cox log-rank test).
the tumor nodule (Fig. 3A, Lane 6) at this time; however, no viral There was no difference in survival between the low and middle dose
DNA was detected in brain, spinal cord, adrenal gland, lung, or liver, animals (P = 0.65).
confirming the immunohistochemistry results. In contrast, wild-type In both of the survival studies, approximately 20% of tumor
HSV led to detectable HSVtk sequences in the brain (Fig. 3B, Lane 1). bearing animals died suddenly 2â€”5days after HSV-l716 administra
Fig. 3B shows the results from two tumor-bearing animals 14 days tion. Because of autolysis, only limited autopsies could be performed,
after i.p. injection of HSV-17l6. Again, no viral DNA was detected in and the cause of these early deaths remains unknown. These animals
any of the normal tissues assayed. did not display signs of encephalitis (i.e., no ruffled fur, no paralysis,
HSV-1716 Reduces i.p. Tumor Burden and Increases Survival and no ataxia) before their deaths. Immunohistochemistry and PCR
in a SC@ Mouse Model of Human Mesothelioma. To determine data (see above) ruled out widespread viral dissemination. To study
the possibility that early deaths in treated tumor-bearing animals were
the ability of HSV-l7l6 infection to eradicate established tumor,
due to acute renal failure after massive tumor lysis, serum creatinine
animals that had previously received 30 million i.p. REN tumor cells
and uric acid were also studied. The serum creatinine level in a pooled
were given 5 X 106 PFU of HSV-1716 by i.p. injection. At the time
specimen from four tumor-bearing animals 3 days after HSV-l716
of viral administration, animals had established i.p. tumors consisting
administration was 0.2 mg/dl, and the uric acid level was 4.0 mg/dl.
of multiple 5â€”8-mmnodules with portal invasion and gall bladder
The serum creatinine level in matched untreated tumor-bearing mice
distention. Two weeks later, animals were sacrificed, and the tumor
was 0.3 mg/dl, and the uric acid level was 3.9 mg/dl.
burden was assessed using a previously developed semiquantitative
scale that accounts for both gross and microscopic tumor (23). The
tumor score ranges from 0 (no gross or microscopic tumor) to a DISCUSSION
maximum score of 4.0. There was a significant reduction in the mean In this report, we demonstrate that the mutant â€œreplication-restrict
tumor score at day 28 in tumor-bearing animals (n = 12) treated with H
edâ€• SV-1716 (an ICP 34.5 null mutant) can reduce tumor burden
HSV-l716 compared with the mean tumor score in control animals and significantly prolong survival in an animal model of localized
(n 8). The mean tumor score in the treatment group was 1.4 Â±0.2 non-CNS human malignancy. Furthermore, we show that after i.p.
compared with a mean tumor score in the control group of 3.9 Â±0.1 injection of HSV-1716 into SCID mice bearing human tumors, rep
(P < 0.001). All animals in the control group survived the study lication is restricted to the tumor and does not disseminate or replicate
outside the tumor. Although HSV is normally a neurotropic virus,
4 K. Amin, unpublished data. these findings highlight the tumor-killing potential of HSV-l716 for
TREATMENT MAUGNANT W
unknown but are under active investigation in a number of laborato
A ries. The JCP 34.5 gene encodes a protein of 263 amino acids
consisting of a large amino-terminal domain, a linker region of three
@ 1. tkplesmld control amino acid repeats (Ala-Thr-Pro, AlP codons), and a carboxyl
2. breln terminal domain (27). The carboxyl-terminal domain is homologous
to domains of MyD1 16 and growth arrest and DNA damage gene 34
3. spInalcord (GADD 34; Refs. 28 and 29), cellular genes involved in cell cycle
4. lIver regulation and programmed cell death. Recent work suggests that the
HSV ICP 34.5 gene precludes cells from triggering total protein
5. adrenal synthesis characteristic of programmed cell death, at least in a neu
roblastoma system (30). However, this does not appear to be a
, - 6. tumor universal response, and at this time all that can be said is, that in
@ lung contrast to normal cells, malignant cells have abnormalities in cell
cycle and/or protein synthesis pathways that appear to render them
6. DRG permissive for viruses lacking the ICP 34.5 gene. We have recently
PCR demonstrated in a range of primary human glioblastoma cells, that
permissivity for HSV-1716 replication correlates positively with the
rapidly dividing nature of the cells (26).
S 1. 17+breln The ultimate clinical utility of replication-restricted viruses such as
HSV-1716 will be determined by the balance between efficacy and
. 2. PCR water control
safety. The experiments presented here, and by others in brain tumor
models, indicate that mutant herpes viruses can selectively replicate in
3. BraIn human and murine malignant cells to induce the regression of estab
lished tumors. Two factors inherent in an HSV-based approach to
4. lungs mesothelioma therapy provide potentially valuable safety features. (a)
@ 5. heart Animal 1 The retention of the HSVtk gene in HSV-l716 will allow treatment
with the antiviral drugs acyclovir or ganciclovir at any point to abort
â€˜ 6. lIver the infection. (b) Administration of virus to a peripheral location, such
as the pleural or peritoneal cavity rather than directly into the brain,
7. ovary should increase the margin of safety.
8. braIn One issue that will likely affect the efficacy of replication-restricted
HSV mutants will be the immune response of the host to virus. A
critical balance between optimal viral replication leading to tumor
9. heart Animal 2 destruction and the immune-mediated elimination of viral infection
10. lungs must be achieved. The efficacy of replication-restricted HSV infection
11. lIver in immunocompetent animals with non-CNS malignancy, and even
more importantly in immunocompetent HSV-primed animals, will
@& 12. ovary
need to be analyzed.
Safety issues, specifically the possibility of viral dissemination
Fig. 3. A, PCR using primers for the HSVtk gene were performed on a control plasmid
containing the HSVtk gene (lane 1) and from DNA extracted from tissues in a tumor
bearing animal 3 days after i.p. HSV-1716 virus administration(Lanes2â€”8). he Southern
blot demonstrates HSVtk signal in the tumor nodule and lack of signal in normal tissues.
DRG, dorsal root ganglion. B, PCR using primersfor the HSVtk gene were performed .8
from DNA extracted from the brain of an animal 5 days after i.p. injection of wild-type
HSV-l7 (Lane 1), from tissues of tumor-bearing animals 14 days afteri.p. virus admin x Contxol
istration(Lanes3â€”12), on a controlplasmidcontaining HSVtkgene(Lane13). U HighDoseVirus
The Southern blot demonstrates the presence of viral HSVtk in the brain of the animal I .6 A
Mi@ Dose Virus
infected with wild-type virus. In contrast, there is no HSV DNA detected in tissues from
animals treated with HSV-l7I6.
localized malignancies of nonneuronal origin such as malignant me
sotheioma. In addition, we found that a variety of nonneuronal tumor
cell types are susceptible to HSV lysis in vitro, but that susceptibility
is not universal. Recently, we have shown that some cell lines derived
from tumors of the CNS can also be essentially resistant to lyric 0
infection by wild-type HSV and/or ICP 34.5 null mutants (26). We are 0 10 20 30 40 50 60 70 80 90 100110120130140150160170180
presently screening a large number of tumors of both neuronal and
Time post tumorcell injection(days)
nonneuronal lineage with the goal of better defining the cellular
factors that restrict lyric HSV infection. Fig. 4. HSV-l716 viral dose-response survival study. SC@ mice received 3 X l0@
The â€œreplication-restrictionâ€• marked attenuation of neuroviru
and human malignant mesothelioma cells on day 0. Seven days later, one animal was
sacrificed to confirm tumor. The remaining animals were randomized into four groups:
lence of HSV-1716 results from deletion of the gene coding for ICP control(n = ll,culturemedia),lowdose(n = 10,5 X lO@PFUHSV-17l6),middledose
34.5. The precise mechanisms responsible for these properties are still (n 10, 5 X 10' PFU HSV-1716), and high dose (n = 10, 5 X 1O@ PFU-l7l6).
TREATMENT MALIGNANT WITHHSV
resulting in CNS infection with or without neural latency, will also on the use of viruses in the treatment of carcinoma of the cervix. Cancer (Phila.), 9:
determine the ultimate clinical utility of replication-restricted viruses
12. MacLean, A. R., Ul-Fareed, M., Robertson, L., Harland, J., and Brown, S. M. Herpes
such as HSV-1716. Administration of HSV-1716 to the peritoneal simplex virus type 1 deletion variants 1714 and 1716 pinpoint neurovirulence-related
cavity did not result in detectable viral dissemination as evaluated by sequences in Glasgow strain 17+ between immediate early gene 1 and the â€œaâ€•
sequence. J. Gen. Virol, 72: 631â€”639, 1991.
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